Transformation of microorganisms and cultured cells using genetic engineering is currently applied to the production of physiologically active substances useful as medicines and thus greatly contributes to the industry. In the field of plant breeding, industrial application of genetic engineering lags behind because the life cycles of plants are much longer than those of microorganisms. However, since this technology enables a desired gene to be directly introduced into plants to be bred, it has the following advantages compared to classical breeding which requires multiple crossing.
(a) It is possible to introduce only a characteristic to be improved.
(b) It is possible to introduce characteristics of species other than plants (such microorganisms and the like).
(c) It is possible to greatly shorten the breeding period.
Thus engineering methods for plant breeding have been investigated vigorously.
The production of transgenic plants requires the following three steps.
(1) Introducing the desired gene into the plant cell (including introduction of the same into the chromosomes, nucleus and the like).
(2) Selecting plant tissue made only of cells into which the desired gene has been introduced.
(3) Regenerating a plant from the selected plant tissue.
In order to select transgenic tissues into which a desired gene has been introduced, visual identification of the tissue in which the desired gene is expressed without regenerating the new plant has been desired. To achieve this, the desired gene is typically introduced into the plant cell together with a marker gene of which the expression can be easily detected at the stage of cultivating the cell. That is, the expression of the marker gene is used as an index of the introduction of the desired gene. Examples of conventional marker genes include antibiotics-resistant genes, such as kanamycin-resistant gene (i.e., NPTII; neomycin phosphotransferase gene), a hygromycin-resistant gene (i.e., HPT; hygromycin phosphotransferase gene), amino acids synthetase genes, such as a nopaline synthetase gene (NOS), an octopine synthetase gene (OCS), and a sulfonylurea-resistant gene (i.e., ALS; acetoactate synthetase gene) that imparts agricultural chemical resistance.
However, the expression of a marker gene can case serious problems when such a transgenic plant is used for food. That is, it is quite difficult to ensure that a gene product produced by expressing a marker gene is safe for the human body. Consequently, if a transgenic plant containing a marker gene is to be sold as a food, a detailed investigation must be performed to determine the influence of the marker gene on the human body. For example, the NPTII gene has been used as a marker gene at the laboratory level since the early 1980s. In 1994, the product of that gene was finally accepted as a food additive by the U.S. Food and Drug Administration (FDA). Since then, transgenic plants containing the NPTII gene as a marker gene have been used for food. However, some consumers of products containing the NPTII gene are still anxious about this gene's effect.
Moreover, marker gene which are practically used are only genes, such as the NPTII gene, which contribute to detoxification of a growth inhibitory substance in plant cells. Therefore, to select transgenic plant tissue into which a desired gene has been introduced, the tissue is cultivated in a culture medium containing the growth inhibitory substance, and the expression of the marker gene, namely the resistance at the tissue to the growth inhibitory substance is evaluated and used as an index. However, even when a tissue has such a resistance, cultivation in the presence of an inhibitory substance can result in undesirable side effects on the plant cells, such as a decrease in proliferation and redifferentiation of the transgenic tissue.
Further, the expression of a marker gene in a plant cell after the selection of transgenic tissue seriously obstructs plant breeding by subsequent gene introduction. That is, when another gene is introduced into a transgenic plant containing a marker gene, the gene introduction must be monitored using a different marker gene. However, the effectiveness of a marker gene various with the plant species. Therefore, a preliminary test is required to set the conditions for each marker gene (for example, it is reported that the HPT gene is more effective in rice plants than the NPTII gene (K. Shimamoto et al., Nature (London), vol.338, p.274, 1989)). Still further, since the varieties of marker gene are limited, the multiple introduction of a gene cannot be repeated indefinitely simply by changing the marker gene. That is, the number of gene introductions into a certain plant is limited itself by the variety of marker genes which can be used in that plant. Besides, the kind of the marker gene which can be actually used is limited as mentioned above. Accordingly, it is desirable to find a method for removing the marker gene from the chromosome after selection of the transgenic plant tissue to exclude the influence of the marker gene from the cell, tissue and plant.
To eliminate the influence of a marker gene, two methods have been reported. In one method, a marker gene and a transposon of the plant is introduced into a plant chromosome and subsequently removed therefrom following transposon (International Laid-Open No. WO 92/01370). In a second method, the site-specific recombination system of P1 phage is used instead of the transposon (International Laid-Open No. WO 93/01283). Using these methods, it is possible to obtain a cell in which the marker gene has been removed from the plant chromosome at a given ratio after the introduction of the gene. Unfortunately, the probability that the marker gene is removed is very low.
Further, plant cells in which the marker genes have been removed from the chromosomes using these methods are scattered among the cells in which the marker genes are still present and expressed. These two kinds of the cells cannot be distinguished visually.
Plant cells containing marker genes and a desired gene can be selected based on their chemical resistance, nutritional requirements and the like. However, at the time of the selection, the cells lacking marker genes exhibit serious growth inhibition and are destroyed in many cases. Accordingly, these selections cannot be applied to obtain cells lacking marker genes.
In order to obtain plants which lack a marker gene and which contain the desired gene using the above-mentioned methods, the tissue of plant, in which cells lacking the marker gene and cells containing the marker genes are mixed, are proliferated, regenerated, and then analyzed for the selection, using methods such as Southern hybridization or polymerase chain reaction. This method is based on the premise that a regenerated individual is derived from a single cell and therefore all of the plant's cells should have the same characteristics. Thus, an individual derived from a cell lacking the marker gene are made only of such cells. Unfortunately, cells constituting such a regenerated individual are not necessarily uniform. Cells lacking the marker gene chromosome and cells containing the marker gene are coexistent and distributed quite irregularly even in the same individual regenerated plant and in the same tissue thereof. Thus, it is extremely difficult to obtain an individual made only of cells lacking the marker gene at the stage in which the cultured tissue is redifferentiated to regenerate the individual.
In addition, known analytical methods of selection use a tissue, such as a leaf, as a test sample (not a whole individual or a single cell). Consequently, only the overall tendency is analyzed with respect to the state of the marker gene present in one leaf. Besides, in this case, it is common that the marker gene-free cell and the marker gene-containing cell are both present in the same individual or tissue. So, even if an individual made only of cells lacking the marker gene happens to be formed, it is difficult to select this. Even if the presence of the marker gene is not detected in this tissue, tissues in other sites of the same individual may contain the marker gene, or it simply shows that the amount of the marker gene is below the detected limit. Therefore, it is impossible to determine if the test sample is completely free from the marker-gene-containing cells.
Using the above-mentioned methods, an individual lacking the marker gene is obtained only from a germ cell, such as a pollen, an egg cell and the like. When the self-pollination is conducted using the egg cell lacking the marker gene, a fertilized egg lacking the marker gene is obtained at a fixed ratio according to a classical hereditary law, and from this fertilized egg, an individual made only of cells having the same characteristics as the fertilized egg is produced. Conventional analytical methods such as Southern hybridization may be conducted using this individual. Namely, even if the cell lacking the marker gene is produced by the method described in the report referred to here, the individual made only of such a cell is obtained for the first time by redifferentiating the plant from the cultured tissue containing such a cell, conducting crossing of the regenerated plant and obtaining progeny of F.sub.1 or later generations. The thus-obtained individual can be selected as an individual lacking the marker gene.
In order to remove the marker gene from the transgenic plant, JP-A-6-276872 reports a technique for gene introduction in which a marker gene is inserted into a separate plasmid vector different from the vector containing the desired gene. The plasmid containing the marker gene is removed from the cell after the completion of the gene introduction (the term "JP-A" as used herein means a Japanese published patent application). However, this technique requires a crossing step for the removal of the marker gene. In this respect, the technique is the same as those of the above-mentioned two reports.
The above methods are difficult to apply to woody plants that have a long growth period, sterile individuals or hybrid individual in which F.sub.1 is itself valuable. Further, when removable DNA elements, such as a transposon and the like, are used, the ratio at which these elements are removed from the chromosomal DNA, virus vector DNA and the like where these elements are present and function is typically extremely low. Accordingly, it is necessary that the removal of these elements (namely, the removal of the marker gene) can be easily detected actually at least at the stage of the cultured tissue. When this cannot be detected before redifferentiation of the cultured tissue and the formation of a later generation via the crossing of the regenerated individual, the method is impractical.